Each year, infectious diseases kill more than 17 million people, including 9 million children. In the United States, deaths due to infectious diseases increased 58 percent from 1982 to 1992 and are now third in the leading causes of death. In addition to suffering and death, infectious diseases impose an enormous financial burden on society. The majority of those infections and deaths are caused by organisms that first make contact with and then either colonize or cross mucosal surfaces to infect the host.
While vaccination is the most cost-effective means of controlling infectious disease morbidity and mortality, traditional vaccine strategies that involve parenteral immunization (via needle) with inactivated viruses or bacteria or subunits of relevant virulence determinants of those pathogens do not prevent those interactions. In fact, traditional vaccine strategies do not prevent infection but instead resolve infection before disease ensues. In some cases, HIV for example, once the virus crosses the mucosal surface and enters the host cell, be that a dendritic cell, an epithelial cell, or a T-cell, the host-parasite relationship is moved decidedly in favor of the parasite (HIV). In that case, as in many others, a vaccine strategy that does not prevent the initial infection of the host is unlikely to succeed.
Recently, a great deal of attention has focused on mucosal immunization as a means of inducing secretory IgA (S-IgA) antibodies directed against specific pathogens of mucosal surfaces. The rationale for this is the recognition that S-IgA constitutes greater than 80% of all antibodies produced in mucosal-associated lymphoid tissues in humans and that S-IgA may block attachment of bacteria and viruses, neutralize bacterial toxins, and even inactivate invading viruses inside of epithelial cells. In addition, the existence of a Common Mucosal Immune System permits immunization on one mucosal surface to induce secretion of antigen-specific S-IgA at distant mucosal sites. It is now appreciated that mucosal immunization may be an effective means of inducing not only S-IgA but also systemic antibody and cell-mediated immunity.
The mucosal immune response can be divided into two phases (McGhee and Kiyono 1993, Infect. Agents. Dis. 12:55-73). First, the inductive phase involves antigen presentation and the initiation events which dictate the subsequent immune response. During the initiation events, antigen-specific lymphocytes are primed and migrate from the inductive sites (e.g., Peyer's patches in the enteric mucosa) through the regional lymph nodes, into the circulation and back to mucosal effector sites (e.g. lamina propria). Once these effector cells have seeded their effector sites, the second phase, or effector phase, of the mucosal immune response can occur. A significant difference between mucosal immunization and parenteral immunization is that both mucosal and systemic immunity can be induced by mucosal immunization while parenteral immunization generally results only in systemic responses.
Most studies on the mucosal immune response conducted to date have dealt with the secretory antibody component of the mucosal response and the complex regulatory issues involved with induction of S-IgA following mucosal immunization and not with the systemic antibody response or cellular immunity induced by mucosal immunization. In that regard, it is important to understand the type of helper T lymphocyte response induced by mucosal immunization since the type of helper T lymphocyte stimulated by an antigen is one of the most important factors for defining which type of immune response will follow. At least two different types of helper T lymphocytes (Th) which can be distinguished based on cytokine secretion have been identified in mice (Cherwinski et al. 1987, Journal of Experimental Medicine 166:1229-1244; Mosmann and Coffman 1989, Annual Reviews of Immunology 7:145-173), humans (Romagnani 1991, Immunology Today 12:256-257) and other animal species (Brown et al. 1994, Infection and Immunity 62:4697-4708). Th1 lymphocytes secrete substantial amounts of IL-2 and IFN-gamma and execute cell-mediated immune responses (e.g. delayed type hypersensitivity and macrophage activation), whereas Th2 lymphocytes secrete IL-4, IL-5, IL-6 and IL-10 and assist in antibody production for humoral immunity. Theoretically then, antigenic stimulation of one T helper cell subset and not the other would result in production of a particular set of cytokines which would define the resulting immune response.
The presence of these cytokines coupled with an antigenic stimulus presented by macrophages in the context of Class II MHC molecules can initiate a Th1 type responses. The ability of Th1 cells to secrete IL-2 and IFN-gamma further amplifies the response by activating Th1 cells in an autocrine fashion and macrophages in a paracrine fashion. These activated leukocytes can release additional cytokines (e.g., IL-6) which may induce the proliferation and differentiation of antigen specific B lymphocytes to secrete antibody (the effector phase). In this scenario, the predominant isotype secreted by murine B lymphocytes is often IgG2a. In a second scenario (Urban et al. 1992, Immunol. Rev. 127:205-220), antigens such as allergens or parasites can effectively stimulate a Th2 lymphocyte response (the inductive phase). Presentation of such antigens to Th2 cells can result in the production of the lymphokines IL-4 and IL-5 which can induce antigen specific B lymphocytes to secrete IgE and IgG1 or induce eosinophilia, respectively (the effector phase). Furthermore, stimulated Th2 cells can secrete IL-10 which has the ability to specifically inhibit secretion of IL-2 and IFN-gamma by Th1 lymphocytes and also to inhibit macrophage function.
It is obvious that the type of T helper cell stimulated affects the resultant cellular immune response as well as the predominant immunoglobulin isotype secreted. Specifically, IL-4 stimulates switching to the IgE and IgG1 isotypes whereas IFN-gamma stimulates IgG2a secretion. Numerous studies, predominantly conducted in vitro, have suggested that IL-5, IL-6 and TGF-beta can cause isotype witching to IgA.
2.1. Bacterial Enterotoxins as Mucosal Adjuvants
Despite the attractiveness of mucosal vaccination for inducing both mucosal and systemic immune responses, mucosally administered antigens are frequently not immunogenic. A number of strategies have been developed to facilitate and enhance the immune response obtained after mucosal immunization. Among these strategies are the use of attenuated mutants of bacteria (i.e., Salmonella spp.) as carriers of heterologous antigens, encapsulation of antigens into microspheres, gelatin capsules, different formulations of liposomes, adsorption onto nanoparticles, use of lipophilic immune stimulating complexes, and addition of bacterial products with known adjuvant properties. While a number of substances of bacterial origin have been tested as mucosal adjuvants (Lowell et al. 1997, Journal of Infectious Diseases 175:292-301; Roberts et al. 1995, Infection and Immunity 63:2100-2108; Van De Verg et al. 1996, Infection and Immunity 64:5263-5268), the two bacterial proteins with the greatest potential to function as mucosal adjuvants are cholera toxin (CT), produced by various strains of Vibrio cholerae, and the heat-labile enterotoxin (LT) produced by some enterotoxigenic strains of Escherichia coli (Clements et al. 1988, Vaccine 6:269-277; Elson 1989, Immunology Today 146:29-33; Lycke et al. 1992, European Journal of Immunology 22:2277-2281; Xu-Amano et al. 1993, Journal of Experimental Medicine 178:1309-1320).
Although LT and CT have many features in common, these are clearly distinct molecules with biochemical and immunologic differences which make them unique (see below). Both LT and CT are synthesized as multisubunit toxins with A and B components. On thiol reduction, the A component dissociates into two smaller polypeptide chains. One of these, the A1 piece, catalyzes the ADP-ribosylation of the stimulatory GTP-binding protein (GSa) in the adenylate cyclase enzyme complex on the basolateral surface of the epithelial cell resulting in increasing intracellular levels of cAMP. The resulting increase in cAMP causes secretion of water and electrolytes into the small intestine through interaction with two cAMP-sensitive ion transport mechanisms involving 1) NaCl cotransport across the brush border of villous epithelial cells, and 2) electrogenic Na dependent Cl secretion by crypt cells (Field, 1980, In: Field M, Fordtran J S, Schultz S G, editors. Secretory diarrhea. Baltimore, Md.: Waverly Press. p 21-30). The B-subunit binds to the host cell membrane receptor (ganglioside GM1) and facilitates the translocation of the A-subunit through the cell membrane.
Recent studies have examined the potential of CT and LT to function as mucosal adjuvants against a variety of bacterial and viral pathogens using whole killed organisms or purified subunits of relevant virulence determinants from these organisms. Representative examples include tetanus toxoid (Xu-Amano et al. 1994, Vaccine 12:903-911; Xu-Amano et al. 1993, Journal of Experimental Medicine 178:1309-1320; Yamamoto et al. 1997a, Proceedings of the National Academy of Sciences 94:5267-5272; Yamamoto et al. 1997b, Journal of Experimental Medicine 185:1203-1210), inactivated influenza virus (Gluck et al. 1999, J Virol 73(9):7780-6; Hashigucci et al. 1996, Vaccine 14:113-119; Katz et al. 1996, In: Brown L E, Hampson A W, Webster R G, editors. Options for the control of influenza. III. New York: Elsevier Science. p 292-297; Katz et al. 1997, Journal of Infectious Diseases 175:352-363; Komase et al. 1998, Vaccine 16(2-3) :248-254), recombinant urease from Helicobacter spp. (Lee et al. 1995, Journal of Infectious Diseases 172:161-171; Weltzin et al. 1997, Vaccine 4:370-376), pneumococcal surface protein A from Streptococcus pneumoniae (Wu et al. 1997, Journal of Infectious Diseases 175:839-846), Norwalk virus capsid protein (Mason et al. 1996, Proceedings of the National Academy of Sciences 93:5335-5340), synthetic peptides from measles virus (Hathaway et al. 1995, Vaccine 13:1495-1500), and the HIV-1 peptides (Staats et al. 1996, Journal of Immunology 157:462-472). There are many other examples and it is clear from these studies that both LT and CT have significant potential for use as adjuvants for mucosally (and otherwise) administered antigens. This raises the possibility of an effective immunization program against a variety of pathogens involving the administration of killed or attenuated organisms or relevant virulence determinants of specific agents in conjunction with LT or CT, preferably mucosally. However, the fact that these toxins stimulate a net lumenal secretory response may prevent their use for practical vaccine applications. For instance, it was observed that as little as 5 μg of purified CT administered orally was sufficient to induce significant diarrhea in human volunteers while ingestion of 25 μg of CT elicited a full 20-liter cholera purge (Levine et al. 1983, Microbiological Reviews 47:510-550).
In recently conducted volunteer studies with LT administered alone or in conjunction with the V. cholerae Whole Cell/B-Subunit Vaccine, LT was shown to induce fluid secretion at doses as low as 2.5 μg when administered in conjunction with the vaccine, while 25 μg of LT elicited up to 6-liters of fluid secretion. While the adjuvant effective dose in humans for either of these toxins has not been established, experiments in animals suggest that it may be comparable to the toxic dose. Taken together, these studies suggest that while LT and CT may be attractive as adjuvants, studies in animals do not reflect the full toxic potential of these molecules in humans, and that toxicity may seriously limit their practical use.
2.2. Differences Between CT and LT
As mentioned above, although LT and CT have many features in common, these are clearly distinct molecules with biochemical and immunologic differences which make them unique (Dickinson and Clements, 1996, In: Kiyono H, Ogra P L, McGhee J R, editors. Mucosal Vaccines. San Diego, Calif.: Academic Press. p 73-87). For example, LT has an unusual affinity for carbohydrate containing matrices (Clements and Finkelstein 1979, Infection and Immunity 24:760-769; Clements et al. 1980, Infection and Immunity 24:91-97). LT binds not only to agarose in columns used for purification but, more importantly, to other biological molecules containing galactose, including glycoproteins and lipopolysaccharides. This lectin-like binding property of LT results in a broader receptor population on mammalian cells for LT than for CT which binds only to GM1 (Angstrom et al. 1994, Proc Natl Acad Sci U S A 91(25):11859-63; Clements et al. 1980, Infection and Immunity 24:91-97; Holmgren, 1994, Progress in Brain Research 101:163-177). Moreover, LT and CT generally activate different subsets of T helper cells. CT promotes CD4+ Th2-type responses and help for IgG1, IgE and mucosal IgA while LT induces both CD4+ Th1- and Th2-type responses and help for IgG1, IgG2a, IgG2b, and mucosal IgA (Marinaro et al. 1995, Journal of Immunology 155:4621-4629; Xu-Amano et al. 1993, Journal of Experimental Medicine 178:1309-1320). This distinction between LT and CT may be important in terms of selecting a mucosal adjuvant for use with specific categories of pathogens, assuming the Th2 bias holds. Possible sources for this bias include the availability of different receptors for LT and CT, mentioned above, differences in intracellular localization based upon differences in ER-signal sequences between CT and LT, and differences in activation of intracellular signaling pathways. CT, LT strains obtained from human hosts, and LT strains obtained from porcine hosts have -KDEL, -RNEL, and -RDEL, respectively, as ER retention signals.
2.3. Development of Non-Toxic Mucosal Adjuvants
A number of attempts have been made to alter the toxicity of LT and CT, most of which have focused on eliminating enzymatic activity of the A-subunit associated with enterotoxicity. The majority of these efforts have involved the use of site-directed mutagenesis to change amino acids associated with the crevice where NAD binding and catalysis is thought to occur. Recently, a model for NAD binding and catalysis was proposed (Domenighini et al. 1994, Molecular Microbiology 14:41-50; Pizza et al. 1994, Molecular Microbiology 14:51-60) based on computer analysis of the crystallographic structure of LT (Sixma et al. 1993, Journal of Molecular Biology 230:890-918; Sixma et al. 1991, Nature (London) 351:371-377). Replacement of any amino acid in CT or LT involved in NAD-binding and catalysis by site-directed mutagenesis has been shown to alter ADP-ribosyltransferase activity with a corresponding loss of toxicity in a variety of biological assay systems (Burnette et al. 1991, Infection and Immunity 59:4266-4270; Fontana et al. 1995, Infection and Immunity 63:2356-2360; Harford et al. 1989, European Journal of Biochemistry 183:311-316; Hase et al. 1994, Infection and Immunity 62:3051-3057; Lobet et al. 1991, Infection and Immunity 59:2870-2879; Lycke et al. 1992, European Journal of Immunology 22:2277-2281; Merritt et al. 1995, Nature Structural Biology 2:269-272; Moss et al. 1993, Journal of Biological Chemistry 268:6383-6387; Pizza et al. 1994, Molecular Microbiology 14:51-60; Tsuji et al. 1991, FEBS Letters 291:319-321; Tsuji et al. 1990, Journal of Biological Chemistry 265:22520-22525; Yamamoto et al. 1997a, Proceedings of the National Academy of Sciences 94:5267-5272; Yamamoto et al. 1997b, Journal of Experimental Medicine 185:1203-1210). The adjuvanticity potential of some of these mutants has been tested on animal models using a variety of coadministered antigens (DiTommaso et al. 1996, Infection and Immunity 64:974-979; Lycke et al. 1992, European Journal of Immunology 22:2277-2281; Partidos et al. 1996, Immunology 89:483-487; Yamamoto et al. 1997a, Proceedings of the National Academy of Sciences 94:5267-5272; Yamamoto et al. 1997b, Journal of Experimental Medicine 185:1203-1210). In addition, it has been shown that exchanging K for E112 in LT not only removes ADP-ribosylating enzymatic activity, but cAMP activation and adjuvant activity as well (Lycke et al. 1992, European Journal of Immunology 22:2277-2281). A logical conclusion from the Lycke et al. studies is that ADP-ribosylation and induction of cAMP are essential for the adjuvant activity of these molecules. As a result, a causal linkage has been established between adjuvanticity and enterotoxicity. That is, the accumulation of cAMP responsible for net ion and fluid secretion into the gut lumen was thought to be a requisite to adjuvanticity (see below).
Dickinson and Clements (Dickinson and Clements, 1995, Infection and Immunity 63:1617-1623) explored an alternate approach to dissociation of enterotoxicity from adjuvanticity. Like other bacterial toxins that are members of the A-B toxin family, both CT and LT require proteolysis of a trypsin sensitive bond to become fully active. In these two enterotoxins, that trypsin sensitive peptide is subtended by a disulfide interchange that joins the A1 and A2 pieces of the A-subunit. In theory, if the A1 and A2 pieces cannot separate, A1 may not be able to find its target (adenylate cyclase) on the basolateral surface or may not assume the conformation necessary to bind or hydrolyze NAD.
Dickinson and Clements constructed a mutant of LT using site-directed mutagenesis to create a single amino acid substitution within the disulfide subtended region of the A subunit separating A1 from A2. This single amino acid change altered the proteolytically sensitive site within this region, rendering the mutant insensitive to trypsin activation. The physical characteristics of this mutant were examined by SDS-PAGE, its biological activity was examined on mouse Y-1 adrenal tumor cells and Caco-2 cells, its enzymatic properties determined in an in vitro NAD:agmatine ADP-ribosyltransferase assay, and its immunogenicity and immunomodulating capabilities determined by testing for the retention of immunogenicity and adjuvanticity. This mutant LT, designated LT(R192G), has been shown to be an effective mucosal adjuvant and has recently been evaluated in a series of Phase I safety studies. LT(R192G) is the subject of U.S. Pat. No. 6,019,982 (Mutant enterotoxin effective as a non-toxic oral adjuvant). Clements also constructed a double-mutant LT, LT(R192G/L211A), which has even further reduced toxicity (U.S. Pat. No. 6,033,673).
Tsuji et al. (Tsuji et al. 1997, Immunology 90:176-182) recently demonstrated that a protease-site deletion mutant LT(Δ192-194) also lacks in vitro ADP-ribosylagmatine activity, has a ten-fold reduction in enterotoxicity in rabbit ligated ileal loops, and a 50% reduction and delayed onset of cAMP induction in cultured myeloma cells. LT(Δ192-194) was shown to have increased adjuvant activity for induction of serum IgG and mucosal IgA against measles virus when compared to native LT, LT-B, or LT(E112K). LT(Δ192-194) was effective when administered intranasally, subcutaneously, intraperitoneally, or orally, although mucosal IgA responses were only demonstrated following mucosal administration. These investigators also demonstrated increased adjuvant activity for mucosally administered LT(Δ192-194) in conjunction with KLH, BCG, and Ova.
Other mutants have also been created and tested. The first of those is the active-site mutant of LT designated LT(S63K) developed by Rappuoli and colleagues (Pizza et al. 1994, Molecular Microbiology 14:51-60) and the second is the CT active-site mutant CT (S61F) developed by McGhee and colleagues (Yamamoto et al. 1997a, Proceedings of the National Academy of Sciences 94:5267-5272; Yamamoto et al. 1997b, Journal of Experimental Medicine 185:1203-1210). LT(S63K) was one of a group of LT A-subunit mutants shown to be devoid of biological activity on mouse Y-1 adrenal tumor cells and to lack detectable in vitro ADP-ribosyltransferase activity. LT(S63K) has been shown to be able to enhance production of anti-Ovalbumin (Ova) IgG in the sera and, to a lesser extent, anti-Ova IgA in vaginal secretions of mice immunized intranasally with up to five immunizations consisting of Ova combined with LT(S63K) (DiTommaso et al. 1996, Infection and Immunity 64:974-979). One publication (Partidos et al. 1996, Immunology 89:483-487) and a number of abstracts presented at various scientific meeting have further characterized this molecule as having intranasal adjuvanticity when administered with other antigens. Oral adjuvanticity for LT(S63K) has not been clearly established.
In two recent papers by Yamamoto et al. (Yamamoto et al. 1997a, Proceedings of the National Academy of Sciences 94:5267-5272; Yamamoto et al. 1997b, Journal of Experimental Medicine 185:1203-1210), mutants of CT with mutations in the NAD binding site that lack detectable in vitro ADP-ribosyltransferase activity and enterotoxicity and fail to induce cAMP accumulation in CHO cells were examined for parenteral and mucosal adjuvanticity. In the first study, these investigators demonstrated that CT(E112K) and CT(S61F) retained adjuvanticity for Ova when administered subcutaneously. CT-B alone did not function as an adjuvant, indicating that some portion of the A-subunit must be present for adjuvant activity. Both mutant CTs induced Ova specific CD4+ T-cell proliferative responses with subsequent production of IL-4, IL-5, IL-6 and IL-10 (Th2 type cytokines) comparable to native CT. Significantly, neither native CT nor the mutant CTs promoted Th1 type cytokine development. Importantly, in these studies CT(E112K) exhibited adjuvant activity whereas LT(E112K) had previously been shown not to possess adjuvant activity (Lycke et al. 1992, European Journal of Immunology 22:2277-2281). One possible explanation for this difference in findings is that CT(E112K) was administered subcutaneously while LT(E112K) was administered orally. Alternatively, this may reflect inherent differences between CT and LT. In the second paper by Yamamoto et al., mice were immunized intranasally with CT(S61F) in conjunction with Ova, tetanus toxoid (TT), or influenza virus. Mice showed antigen-specific increases in serum antibodies as well as significant increases in antigen-specific antibodies in nasal and vaginal washes, saliva and fecal extracts that were comparable to those obtained with native CT. Again, CT-B failed to function as an adjuvant when administered intranasally. Both CT(S61F) and native CT elicited Th2 type cytokine secretion and cytokine mRNAs, but not Th1 type cytokine responses.
2.4. Mechanisms of Adjuvanticity
There are a number of potential cellular targets for these bacterially derived adjuvants and the precise mechanism of action remains to be determined. Clearly, significant efforts have been expended to resolve this (Bromander et al. 1993, Scandinavian Journal of Immunology 37:452-458; Cebra et al. 1986, In: Brown F, Channok R M, Lerner R A, editors. Vaccines 86: New approaches to immunization. Developing vaccines against parasitic, bacterial, and viral diseases: Cold Spring Harbor, N.Y. p 129-133; Clarke et al. 1991, Immunology 72:323-328; Clements et al. 1988, Vaccine 6:269-277; Elson 1989, Immunology Today 146:29-33; Elson and Ealding 1984a, Journal of Immunology 133:2892-2897; Elson and Ealding 1984b, Journal of Immunology 132:2736-2741; Elson et al. 1995, Journal of Immunology 154:1032-1040; Hornquist and Lycke 1993, European Journal of Immunology 23:2136-2143; Lycke et al. 1991; Nedrud and Sigmund 1991, Reg. Immunol. 3:217-222; Snider et al. 1994, Journal of Immunology 53:647-657; Takahashi et al. 1996, Journal of Infectious Diseases 173:627-635; Xu-Amano et al. 1994, Vaccine 12:903-911; Xu-Amano et al. 1993, Journal of Experimental Medicine 178:1309-1320). Several models have been proposed, none of which is completely satisfactory. A summary of these proposed mechanisms is found in the review by Freytag and Clements (Freytag and Clements, 1999, Curr Top Microbiol Immunol 236:215-36). Rather than a single defined mechanism, adjuvanticity should be viewed as an outcome and not an event. It is likely to be some combination of effects that collectively results in the observed outcome known as enhanced immunity or adjuvanticity. It is also important to note that most studies attempting to define the mechanism of adjuvanticity of CT and LT focus on induction of sIgA and Th2 events as the only or most relevant outcomes, ignoring the Th1 induction potential of LT which is likely to be important for protection against intracellular bacterial pathogens and viruses.
2.5. Role of cAMP in Adjuvanticity
The role of cAMP in the adjuvanticity of CT, LT, and mutants of CT and LT remains controversial, in part because different mutants of LT and CT have been evaluated using varying techniques in different laboratories and in part because different routes of administration (i.e., intranasal, oral) have been employed with various antigens. A recent report by Cheng et al. (Cheng et al. 1999, Vaccine 18(1-2):38-49) provided a side-by-side comparison of LT, active-site mutants, the protease-site mutant LT(R192G), and recombinant B-subunit for the ability to induce specific, targeted immunologic outcomes using a single, defined antigen (Tetanus Toxoid) following two different mucosal routes of immunization (intranasal or oral). For this study, these investigators employed the Y-1 Adrenal Tumor Cell assay, a non-polarized Caco-2 cell assay for induction of cAMP, the Patent Mouse assay for enterotoxicity (Guidry et al., Infect. And Immun., 1997, 65(12):4943-4950), and an in vitro antigen restimulation assay on splenic mononuclear cells for determination of Th1 and Th2 type cytokine production. The Patent Mouse assay is a modification of the sealed adult mouse assay of Richardson et al. (Richardson et al. 1984, Infection and Immunity 43:482-486). It is slightly less sensitive than the more traditional Rabbit Ligated Ileal Loop assay, but responds to native LT and CT in a dose dependent fashion. In those studies, following intranasal immunization, both Th1 and Th2 type cellular immune responses to TT differed for the various active site mutants and LT-B and were dependent upon their ability to induce cAMP. For example, despite the fact that LT-B given intranasally can induce serum IgG, it is not able to induce any significant level of T cell response. Furthermore, while all active site mutants examined were able to induce antigen-specific antibody responses when administered intranasally, only native LT, LT(A69G), and LT(R192G), which retained the ability to induce production of cAMP, were able to elicit antigen-specific Th1 and Th2 cytokines following intranasal immunization in combination with TT. As with intranasal immunization, production of both Th1 and Th2-type cytokines following oral immunization was correlated with the ability to induce accumulation of cAMP.
Recently, Giuliani et al. (Giuliani et al. 1998, J Exp Med 187(7):1123-32) compared two active-site mutants, LT(S63K) and LT(A72R), for the ability to function as intranasal adjuvants. In those studies, LT(A72R) which retains some level of enzymatic activity was a better intranasal adjuvant for ovalbumin than LT(S63K) which those authors have reported to lack any detectable enzymatic activity. It remains to be seen whether LT(A72R) induces antigen-specific cytokine responses or functions orally in animals or humans.
Both CT and LT have significant potential to function as mucosal adjuvants for co-administered antigens and to facilitate the development of entire new classes of vaccines for-mucosal delivery. The data reported by Cheng et al. makes it clear that different mutants of LT have different properties that vary depending upon the nature of the mutation and the route of delivery. Specifically, those mutants that retain the ability to induce cAMP elicit quantitatively and qualitatively different responses than do those mutants that lack this function. For induction of antigen-specific antibody responses, it appears that any of the mutants examined in Cheng et al. will suffice if delivered intranasally. Clearly, the best cellular responses are elicited by native LT and mutants that retain some cAMP activity and only those that retain some cAMP activity can elicit Th1 type responses when administered orally. Significant serum antigen-specific IgG responses following oral administration were only observed for these mutants as well, and not for those that lacked the ability to induce cAMP.
2.6. Analysis of Hybrid Toxins
One recent study has employed hybrid toxins to explore the differential toxicity of CT and LT (Rodighiero et al. 1999, J Biol Chem 274(7):3962-9; Rodighiero et al. 1998, Biochem Soc Trans 26(4):S364). In those studies, hybrid toxins were constructed in which the A1 fragment of one toxin was substituted for that of the other (CT-A1:LT-A2REDL/LT-B; LT-A1:CT-A2KDEL/CT-B) as well as hybrids in which the putative ER-retention signal was altered (CT-A1:CT-A2RDEL/CT-B; CT-A1:CT-A2RDEL/LT-B). (Importantly, an LT-A/CT-B hybrid was neither created nor evaluated in these studies). The findings from these hybrid toxin studies are 1) CT-A1:LT-A2REDL/LT-B is less potent than wild type CT at inducing chloride secretion; 2) LT-A1:CT-A2KDEL/CT-B induced chloride secretion at levels comparable to levels induced by wild type CT; 3) all of the constructs containing CT-A1 had equivalent ADP-ribosyltransferase activity; 4) wild type LT and LT-A1:CT-A2KDEL/CT-B had higher activity than native CT, and 5the differences between the toxicities of CT and LT are not a function of differences in the ER retention signals. The third hybrid, CT-A1:CT-A2RDEL/LT-B, induced chloride secretion at levels equivalent to wild type CT.
One other hybrid toxin study has been reported (Takeda et al. 1981, Infect Immun 34(2) : 341-6). In that study, LT-A/CT-B and CT-A/LT-B hybrids were prepared by dissociation:re-association chromatography (i.e., the hybrids were not produced recombinantly from an organism designed to express LT-A and CT-B in the absence of LT-B and CT-A, but, instead, were prepared by isolating wild-type LT and CT holotoxin and dissociating said holotoxins into their substituent subunits and then reassembling a hybrid holotoxin by combining the CT-B subunits with the LT-A subunits). The hybrids were reported to have toxicity similar to that of the parent proteins from which the A subunits were derived.
Citation or identification of any reference in any section of this application shall not be construed as an admission that such reference is available as prior art to the present invention.